Treatment of sepsis and septic shock using ghrelin and growth hormone

ABSTRACT

Methods are disclosed for treating sepsis and septic shock using a combination of ghrelin and growth hormone, and for using ghrelin to reduce organ and tissue injury and improve survival after combined radiation exposure and sepsis.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/003,856, filed on Nov. 20, 2007, the content of whichis hereby incorporated by reference into the subject application.

STATEMENT OF GOVERNMENT SUPPORT

The invention disclosed herein was made with U.S. Government supportunder grant numbers R01 AG028352 and R21AI080536 from the NationalInstitutes of Health. Accordingly, the U.S. Government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods for treating sepsis and septicshock using a combination of ghrelin and growth hormone, in particularin geriatric populations, and to methods of using ghrelin for reducingorgan and/or tissue injury and/or improving survival after radiationexposure combined with sepsis.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inparenthesis. Full citations for these references may be found at the endof the specification immediately preceding the claims. The disclosuresof these publications are hereby incorporated by reference in theirentireties into the subject application to more fully describe the artto which the subject application pertains.

Despite advances in the management of trauma victims, the incidence ofsepsis and septic shock has increased significantly over the past twodecades. It has been estimated that in the United States alone, morethan 750,000 patients develop sepsis and septic shock each year with anoverall mortality rate of 28.6%. Severe sepsis is a common, expensive,and frequently fatal condition, with as many deaths annually as thosefrom acute myocardial infarction. Sepsis is the thirteenth leading causeof death overall in the United States. A recent report indicates thatthe average costs per septic patient are at least $22,100, with annualtotal costs of more than $16 billion nationally. Activated protein C(APC) is the only FDA-approved specific treatment for sepsis, but itsuse is limited to non-surgical adult patients with severe sepsis. APCcannot be used in trauma victims and surgical patients who developsepsis, due to its adverse effects on coagulation. Thus, there is agreat need for a new effective therapy for sepsis, especially surgicalsepsis. The market potential for sepsis treatment is estimated at $10-25billion annually in the United States alone.

SUMMARY OF THE INVENTION

The present invention is directed to methods for treating sepsis and/orseptic shock by administering to a subject a combination of ghrelin andgrowth hormone in amounts effective to treat sepsis and/or septic shock.The methods can be used to prevent and/or reduce physiological effectsof sepsis.

The invention also provides pharmaceutical compositions comprisingghrelin and growth hormone formulated in dosage form for treating sepsisand/or septic shock.

The invention further provides methods of preparing a pharmaceuticalcomposition for treating sepsis and/or septic shock, the methodcomprising formulating ghrelin and growth hormone together in apharmaceutical composition in amounts effective to treat sepsis and/orseptic shock.

The invention also provides methods for reducing organ and/or tissueinjury in a subject and/or improving survival of the subject afterradiation exposure and sepsis comprising administering to the subjectghrelin in an amount effective to reduce organ and/or tissue injuryand/or to improve survival.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Graph showing that aging increases mortality after sepsis andseptic shock induced by lipopolysaccharide (LPS) in rats.

FIG. 2A-2B. Graphs showing that proinflammatory cytokine levels aregreater in aged rats than in young rats after LPS administration. A:Tumor necrosis factor alpha (TNF-α); B: Interleukin-6 (IL-6).

FIG. 3A-3D. Graphs showing that there is greater tissue injury in agedrats than in young rats after LPS administration. A: aspartateaminotransferase (AST); B: alanine aminotransferase (ALT), C: totalbilirubin; and D: lactate.

FIG. 4. Graph showing that an LPS injection induces a greater reductionin circulating ghrelin levels in aged rats than in young rats.

FIG. 5. Graph and photograph of a gel after electrophoresis of RT-PCRproducts showing lower ghrelin receptor gene expression in the brain'sdorsal vagal complex of aged rats than young rats.

FIG. 6A-6B. Graphs showing that ghrelin receptor inhibition by D-SP([D-Arg¹ D-Phe⁵ D-Trp^(7,9) Leu¹¹]-substance P) further increasesLPS-induced TNF-α (A) and IL-6 (B) levels.

FIG. 7A-7C. Graphs showing that ghrelin receptor inhibition by D-SPexacerbates LPS-induced tissue damage. A: AST; B: ALT; C: Lactate.

FIG. 8. Graph showing that IV administration of ghrelin receptorinhibitor D-SP increase mortality of rats after CLP.

FIG. 9A-9D. Graphs showing that administration of growth hormone (GH) incombination with ghrelin reduces the induction of TNF-α and IL-6 andtissue damage in aged rats after LPS administration. A: TNF-α; B: IL-6;C: AST; D: ALT.

FIG. 10A-10C. Graphs showing the effect of ghrelin treatment on plasmalevels of TNF-α(A), IL-6 (B) and high-mobility group box 1 protein(HMGB-1) (C) after cecal ligation and puncture (CLP) in young rats.Effects are also shown on photograph gels in C.

FIG. 11A-11C. Graphs showing the effect of ghrelin treatment on cardiacoutput (CO) (A), stroke volume (SV) (B) and total peripheral resistance(TPR) (C) after CLP in young rats.

FIG. 12A-12C. Graphs showing the effect of ghrelin treatment on bloodflow in the liver (Panel A), kidneys (Panel B) and gut (Panel C) afterCLP in young rats.

FIG. 13A-13C. Graphs showing the effect of ghrelin treatment on plasmalactate (Panel A), creatinine (Panel B) and lung edema (Panel C) afterCLP in young rats.

FIG. 14. Graph showing that ghrelin treatment increases survival ofyoung rats after CLP.

FIG. 15. Graph showing IV administration of ghrelin restores reducedbrain ghrelin levels 20 hours post -CLP in young rats.

FIG. 16. Graph showing that ICV injection of ghrelin reduces serumlevels of TNF-α after LPS injection in young rats.

FIG. 17. Ghrelin administration increases survival rate in an animalmodel of radiation exposure followed by sepsis (Radiation CombinedInjury (RCI)).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for treating sepsis and/or septic shockin a subject comprising administering to the subject a combination ofghrelin and a growth hormone in amounts effective to treat sepsis and/orseptic shock.

Sepsis can be characterized by an inflammatory state caused byinfection. It is a toxic condition resulting from the spread of bacteriaor their products from a focus of infection. Septicemia (infection inthe blood) is a subset of sepsis. Critical forms of sepsis includesevere sepsis with acute organ dysfunction and septic shock withrefractory arterial hypotension. Septic shock can be a life-threateningform of sepsis that typically results from gram-negative bacteria andtheir toxins in the bloodstream.

As used herein, to treat sepsis means to prevent or reduce aphysiological effect of sepsis. Preferably, treatment prevents orreduces serum elevation of one or more of TNF-α, interleukin-6,aspartate aminotransferase, alanine aminotransferase, bilirubin,lactate, creatinine, or high-mobility group box 1 protein. Preferably,treatment prevents or reduces tissue and/or organ injury in the subject.Preferably, treatment improves pulmonary edema. Preferably, treatmentimproves cardiovascular stability, as measured by one or more ofimproved cardiac output, stroke volume, total peripheral resistance, orblood flow. Preferably, the treatment prevents or reduces septic shock.Preferably, treatment improves survival of the subject.

The subject can be a mammal of any age. A preferred subject is a humanat least 50 years of age, more preferably at least 60 years of age, andmost preferably at least 65 years of age.

The invention also provides a pharmaceutical composition comprisingghrelin and growth hormone formulated in dosage form for treating sepsisand/or septic shock.

The invention further provides a method of preparing a pharmaceuticalcomposition for treating sepsis and/or septic shock, the methodcomprising formulating ghrelin and growth hormone together in apharmaceutical composition in amounts effective to treat sepsis and/orseptic shock.

Ghrelin can be any of the forms of ghrelin known in the art. Preferably,ghrelin is human ghrelin. Preferably, ghrelin has the sequenceGSSFLSPEHQRVQQRKESKKPPAKLQPR, where S at position 3 is a n-octanoylatedserine (SEQ ID NO:1) (Kojima et al. 1999), or a fragment, homolog oranalog thereof, where the fragment, homolog or analog has at least 90%of the biological activity of SEQ ID NO:1.

Growth hormone can be any of the forms of naturally occurring orsynthetic growth hormones known in the art, or a pro-hormone thereof.Preferably, growth hormone is a human growth hormone. Variants of humangrowth hormone are known to occur (e.g., U.S. Pat. No. 5,962,411; PCTInternational Publication No. WO 03/042408 A2). Preferred forms ofgrowth hormone include, but are not limited to:

  1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnas lrahrlhqla fdtyqefeea 61 yipkeqkysf lqnpqtslcf sesiptpsnr eetqqksnle llrisllliq swlepvqflr121 svfanslvyg asdsnvydll kdleegiqtl mgrledgspr tgqifkqtys kfdtnshndd181 allknyglly cfrkdmdkve tflrivqcrs vegscgf(SEQ ID NO:2) (DeNoto et al. 1981), or a fragment, homolog or analogthereof, where the fragment, homolog or analog has at least 90% of thebiological activity of SEQ ID NO:2;

  1 fptiplsrlf dnaslrahrl hqlafdtyqe feeayipkeq kysflqnpqt slcfsesipt 61 psnreetqqk snlellrisl lliqswlepv qflrsvfans lvygasdsnv ydllkdleeg121 iqtlmgrled gsprtgqifk qtyskfdtns hnddallkny gllycfrkdm dkvetflriv181 qcrsvegscg f(SEQ ID NO:3), or a fragment, homolog or analog thereof, where thefragment, homolog or analog has at least 90% of the biological activityof SEQ ID NO:3; and

  1 mfptiplsrl fdnaslrahr lhqlafdtyq efeeayipke qkysflqnpq tslcfsesip 61 tpsnreetqq ksnlellris llliqswlep vqflrsvfan slvygasdsn vydllkdlee121 giqtlmgrle dgsprtgqif kqtyskfdtn shnddallkn ygllycfrkd mdkvetflri181 vqcrsvegsc gf(SEQ ID NO:4), or a fragment, homolog or analog thereof, where thefragment, homolog or analog has at least 90% of the biological activityof SEQ ID NO:4.

Preferably, ghrelin has an amino acid sequence at least 90% identical toSEQ ID NO:1, and/or growth hormone has an amino acid sequence at least90% identical to SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

Ghrelin and growth hormone can be prepared using recombinant techniquesknown in the art. Ghrelin can be synthesized using techniques known inthe art.

Preferably, ghrelin and growth hormone are formulated in apharmaceutical composition, either separately or in combination. Thesecompositions can be formulated without undue experimentation foradministration to a mammal, including humans, as appropriate for theparticular application. Additionally, proper dosages of the compositionscan be determined without undue experimentation using standarddose-response protocols. For example, ghrelin may be administered in therange of 1-240 nmol/kg body weight, and growth hormone may beadministered in the range of 1-150 μg/kg body weight.

Accordingly, the compositions designed for oral, lingual, sublingual,buccal and intrabuccal administration can be made without undueexperimentation by means well known in the art, for example with aninert diluent or with an edible carrier. The compositions may beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the pharmaceutical compositions ofthe present invention may be incorporated with excipients and used inthe form of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth or gelatin. Examples of excipients includestarch or lactose. Some examples of disintegrating agents includealginic acid, corn starch and the like. Examples of lubricants includemagnesium stearate or potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring and the like. Materialsused in preparing these various compositions should be pharmaceuticallypure and nontoxic in the amounts used.

The compositions of the present invention can easily be administeredparenterally such as for example, by intravenous, intramuscular,intrathecal or subcutaneous injection. Parenteral administration can beaccomplished by incorporating the compositions of the present inventioninto a solution or suspension. Such solutions or suspensions may alsoinclude sterile diluents such as water for injection, saline solution,fixed oils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents. Parenteral formulations may also includeantibacterial agents such as for example, benzyl alcohol or methylparabens, antioxidants such as for example, ascorbic acid or sodiumbisulfate and chelating agents such as EDTA. Buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose may also be added. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

Rectal administration includes administering the pharmaceuticalcompositions into the rectum or large intestine. This can beaccomplished using suppositories or enemas. Suppository formulations caneasily be made by methods known in the art. For example, suppositoryformulations can be prepared by heating glycerin to about 120° C.,dissolving the composition in the glycerin, mixing the heated glycerinafter which purified water may be added, and pouring the hot mixtureinto a suppository mold.

Transdermal administration includes percutaneous absorption of thecomposition through the skin. Transdermal formulations include patches(such as the well-known nicotine patch), ointments, creams, gels, salvesand the like.

The present invention includes nasally administering to the mammal atherapeutically effective amount of the composition. As used herein,nasally administering or nasal administration includes administering thecomposition to the mucous membranes of the nasal passage or nasal cavityof the patient. As used herein, pharmaceutical compositions for nasaladministration of a composition include therapeutically effectiveamounts of the composition prepared by well-known methods to beadministered, for example, as a nasal spray, nasal drop, suspension,gel, ointment, cream or powder. Administration of the composition mayalso take place using a nasal tampon or nasal sponge.

The methods of the present invention prevent or reduce any physiologiceffect of sepsis, including shock (which in turn affects endothelialcell function, smooth muscle contractility, cardiac output, strokevolume, systemic oxygen delivery, lactic acidosis, hemoconcentration,total peripheral vascular resistance and/or regional blood perfusion),renal function, hepatic function, gut absorptive function, adrenalfunction, insulin responsiveness, altered cytokine (e.g., HMGB1, IL-10,TNF-α, IL-1β and/or IL-6) release, and physiological effects of alteredcytokine release (e.g., inflammation). To evaluate the prevention orreduction of physiologic effects of sepsis, it is preferred thatphysiologic effects that are easily measured are compared before andafter treatment. Examples of these effects are elevation of serum TNF-αlevels, elevation of serum ALT levels, elevation of serum AST levels,elevation of serum lactate, and elevation of serum creatinine. Inpreferred embodiments, the measured physiological effect of the sepsisis elevation of serum TNF-α levels. Determination of shock, or itsdirect effects (e.g., hemoconcentration, peripheral vascular resistance,etc.) is also easily measured and can be utilized.

The reduction in a physiological effect of sepsis might also be effectedon such a physiological effect in the absence of sepsis, affordingadditional therapeutic benefits to the methods disclosed herein. Forexample, the disclosed treatments could be used as therapy inindications that include, but are not limited to, inflammatoryconditions. Accordingly, the invention further provides a method fortreating an inflammatory condition in a subject comprising administeringto the subject a combination of a ghrelin and a growth hormone inamounts effective to treat the inflammatory condition.

The invention also provides a method for reducing organ and/or tissueinjury in a subject and/or improving survival of the subject afterradiation exposure combined with sepsis comprising administering to thesubject ghrelin in an amount effective to reduce organ and/or tissueinjury and/or improve survival. Preferably, administration of ghrelinreduces serum elevation of one or more of TNF-α, interleukin-6,aspartate aminotransferase, alanine aminotransferase, lactate,creatinine, lactate dehydrogenase or myeloperoxidase. In one embodimentof the method, growth hormone is administered in combination withghrelin.

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

Experimental Details

Aging Increases Mortality after Endotoxemia and Sepsis Induced by CecalLigation and Puncture (CLP):

Male Fischer-344 rats (young: 3-month-old; aged: 24-month-old) were usedfor assessing the effect of aging on survival in lipopolysaccharide(LPS)-induced endotoxemia. A bolus injection of LPS (15 mg/kg BW; E.coli 055:B5 in 200-μl normal saline) was given intravenously (IV) andsurvival was monitored for 10 days thereafter. The results indicate thatthe survival rate decreased from 90% in young to 10% in aged animals(FIG. 1; n=10/group). Similarly, the survival rate was markedly reducedby 50% in aged animals after CLP and cecal excision 20 h post-CLP ascompared to young animals (data not shown). Thus, aging increasesmortality in both animal models of sepsis. The CLP model of sepsis isassociated with an increase in plasma levels of LPS (6 fold higher thanthe normal value) (Ertel et al., 1991). Sepsis was induced in maleBalb/C mice by cecal ligation and puncture (CLP) as previously described(Li et al. 2007; Wang et al. 2006; Yang et al. 2002).

Aging Exacerbates the Proinflammatory Response and Worsens Tissue Injuryin Endotoxemia:

To determine whether aging plays an important role in inflammation andtissue injury in response to endotoxemia, LPS (15 mg/kg BW, IV) wasgiven to young (3-month-old) and aged (24-month-old) Fischer-344 rats.At 4 h after LPS injection, plasma levels of proinflammatory cytokinesand tissue injury indicators were measured. While plasma levels of TNF-αand IL-6 increased significantly after LPS injection in young animals,the elevation of these cytokines was much greater in aged animals (FIG.2A-B; means±SE, n=5-7/group). This result indicates that a more severehyperinflammatory response occurs in aged animals in response to LPS.The aged animals were also associated with severe tissue injury at 4 hafter LPS injection, as evidenced by significantly elevated levels ofcirculating aspartate aminotransferase (AST, FIG. 3A), alanineaminotransferase (ALT, FIG. 3B), total bilirubin (FIG. 3C), and lactate(FIG. 3D). The increased levels of ALT, AST, and total bilirubin reflecthepatic injury, and hyperlactatemia reflects a low blood flow conditionor hypoxia.

Plasma Ghrelin is Further Reduced in Aged Animals in Endotoxemia:

Ghrelin is an endogenous ligand for the growth hormone secretagoguereceptor. To determine whether aging and/or LPS have any effects onplasma levels of ghrelin, blood samples were collected at 4 h afteradministration of LPS (15 mg/kg BW) in young and aged male Fischer-344rats. Ghrelin levels were assessed by ELISA. The results indicate thatbasal plasma levels of ghrelin are 79.6% higher in aged as compared toyoung rats (Sham, FIG. 4). At 4 h after LPS injection, plasma ghrelindecreased by only 82.4% in young, but decreased by 97.9% in agedanimals. Plasma levels of ghrelin in aged rats were only 21% of those inyoung rats at 4 h after LPS injection (FIG. 4; this difference isstatistically significant as determined by Student's t-test). Despitethe higher levels of ghrelin in aged animals under normal conditions,LPS has a stronger downregulatory effect on ghrelin production/releasein this age group. Plasma ghrelin levels and gene expression are alsoreduced in CLP-induced sepsis (Wu et al., 2004).

Aging Reduces Ghrelin Receptor Expression and Neuronal Activity in theParasympathostimulatory Nuclei of the Brain Stem in Normal Animals:

To determine whether aging is associated with decreased brain ghrelinreceptor expression and neuronal activity, the dorsal vagal complex(DVC) was isolated from the brain stem of aged and young rats. The DVCcontains several nuclei involving in parasympathetic activity (i.e.,DMN—dorsal motor nucleus of the vagus; NTS—nucleus tractus solitarius;and AP—area postrema), which control the vagus nerve efferent output.Ghrelin receptor gene expression in the DVC and neuronal activity in theDMN were measured by RT-PCR (Wu et al., 2004) and c-fosimmunohistochemistry, respectively. The neuronal expression of c-fos iswidely used as a measure of neuronal activation (Martinez et al., 2002).The results indicate that ghrelin receptor gene expression is decreasedsignificantly in the DVC in aged as compared to young animals (FIG. 5).Thus, aging is accompanied by reduction in both brain ghrelin receptorexpression and neuronal activity in the parasympathostimulatory nucleiunder normal conditions.

Ghrelin Receptor Blockade Exacerbates Inflammatory Responses and OrganInjury in Young Rats after LPS Injection:

To further define the role of ghrelin receptor downregulation inproducing hyperinflammatory responses during the aging process, thespecific and potent ghrelin receptor antagonist [D-Arg¹ D-Phe⁵D-Trp^(7,9) Leu¹¹]-substance P (D-SP) (Asakawa et al., 2003) wasinjected IV in 3-month-old Fischer-344 rats (400 nmol/kg BW) for aperiod of 30 min, starting 1 h before LPS injection (15 mg/kg BW).Plasma levels of TNF-α, IL-6, AST, ALT, and lactate were measured at 4 hafter LPS injection. The results indicate that ghrelin receptorinhibition further increased LPS-induced TNF-α and IL-6 levels (FIG.6A-B). In contrast, administration of D-SP in the absence of LPS did notalter plasma levels of TNF-α and IL-6 (FIG. 6A-B). In addition, LPScaused significant organ injury in young animals with the ghrelinreceptor blockade, as demonstrated by higher levels of plasma AST, ALT,and lactate in young animals (FIG. 7A-C). In an additional experiment,the ghrelin receptor antagonist D-SP (100 nmol/kg BW) was given IV as abolus at 5 h after CLP in 3-month-old male rats, followed by continuousIV infusion of D-SP (600 nmol/kg BW) via a primed mini-pump for 15 h(total dose: 700 nmol/kg BW). Survival was recorded for 10 days. Theresults indicate that IV administration of the ghrelin receptorantagonist further decreased the survival rate from 35% to 15% (FIG. 8;n=20/group). These results, taken together, suggest that thehyperinflammatory response after LPS injection in aged animals is likelymediated by the decreased brain ghrelin receptor activation.

Growth Hormone (GH) Sensitizes Ghrelin's Activity by Increasing theCentral Ghrelin Receptor Expression in Aged Animals:

Although administration of ghrelin significantly decreases circulatingproinflammatory cytokines such as TNF-α and IL-6 in sepsis in younganimals (Wu et al., 2004), the present data show that aging markedlyreduces its anti-inflammatory effect since ghrelin alone no longersignificantly reduces TNF-α and IL-6 in aged animals (FIG. 9A-B). Todetermine whether GH sensitizes ghrelin's anti-inflammatory effects, GHwas administered (25 μg/kg BW, a bolus at 30 min prior to LPS injection)with or without ghrelin (20 nmol/kg BW, a bolus at 30 min prior to LPSinjection) in 24-month-old (aged) rats with LPS injection (15 mg/kg BW).The results indicate that neither ghrelin nor GH alone significantlydecreased plasma levels of TNF-α and IL-6 at 4 h after LPS injection. Incontrast, co-administration of ghrelin and GH markedly reduced TNF-α andIL-6 levels (FIG. 9A-B). Similarly, organ injury indicators such as ASTand ALT were attenuated only following co-administration of ghrelin andGH after LPS injection in aged animals (FIG. 9C-D). Taken together,these results strongly suggest that the upregulatory effect of GH onghrelin receptor is the molecular basis of the GH's sensitizing effecton ghrelin responsiveness.

Protective Effects of Ghrelin in Young Septic Animals:

This study was designed to determine whether ghrelin by itself canattenuate sepsis-induced inflammatory responses, cardiovascularresponses, tissue injury, and mortality in young animals. To study this,male 3-month-old (young) rats were subjected to sepsis by CLP. At 5 hafter CLP, a bolus IV injection of ghrelin (6 nmol/kg BW) was followedby a continuous infusion of ghrelin (40 nmol/kg BW) via a primed 200-μlAlzet mini-pump for 15 h. 20 h after CLP (i.e., severe sepsis), plasmalevels of TNF-α, IL-6, and HMGB-1 were determined Cardiac output (CO),stoke volume (SV), total peripheral resistance (TPR), and organ bloodflow (BF) were measured using radioactive microspheres. Moreover, tissueinjury indicators such as plasma levels of lactate and creatinine, aswell as lung water content (edema) were determined. In additional groupsof animals, the necrotic cecum was excised at 20 h after CLP, and a10-day survival was recorded. The results indicate that ghrelinadministration significantly reduced plasma levels of inflammatorycytokines such as TNF-α (FIG. 10A), IL-6 (FIG. 10B) and HMGB-1 (FIG.10C) in CLP-induced sepsis. Similarly, the cytokine levels in peritonealfluid were also markedly reduced by ghrelin treatment 20 h after CLP(data not shown). In addition, ghrelin treatment reduced TPR, increasedCO, SV (FIG. 11A-C) and organ blood flow in the liver (FIG. 12A),kidneys (FIG. 12B), and gut (FIG. 12C), decreased plasma lactate (FIG.13A), creatinine (FIG. 13B) and lung edema (FIG. 13C), and improvedsurvival of septic animals (FIG. 14, from 35% to 65%, n=20/group). Thus,ghrelin may provide a novel approach for anti-sepsis therapy in theyoung population. Moreover, IV administration of ghrelin restored thereduced brain ghrelin levels 20 h post-CLP (FIG. 15). This also confirmsthat ghrelin can cross the blood-brain barrier to produce its beneficialeffect centrally, thus reducing proinflammatory cytokines in sepsis. Tofurther verify that the anti-inflammatory property of ghrelin is througha direct central nervous system interaction, an intracerebroventricular(ICV) administration of ghrelin (3 nmol/kg BW in 10 μl) was performed 30min prior to the IV injection of LPS (15 mg/kg BW). Plasma levels ofTNF-α were measured at 1 h after LPS injection. The results indicatethat ICV injection of ghrelin reduced serum levels of TNF-α by 65% inendotoxemia (FIG. 16). However, due to the presence of central ghrelinhyporesponsiveness in aged animals, it is most likely that treatmentwith ghrelin alone will not produce desirable beneficial results in thegeriatric population.

Summary of Studies:

Using animal models of LPS-induced endotoxemia and CLP-inducedpolymicrobial sepsis, aging has been shown to be associated withincreased mortality. Aging also exacerbates the proinflammatory responseand worsens tissue injury after LPS injection, as demonstrated byincreased TNF-α, IL-6, and various organ injury indicators. Plasmalevels of ghrelin were dramatically reduced after LPS injection or CLP.Since aging reduces ghrelin receptor expression and neuronal activity inparasympathostimulatory nuclei of the brain, it is postulated that thehyperinflammatory response observed in vivo after LPS injection in agedanimals is caused by a central nervous hyporesponsiveness to ghrelin.This is further supported by the finding that ghrelin receptor blockadeexacerbates inflammatory responses and organ injury in young animalsafter LPS injection. Additional data suggest that GH is aghrelin-sensitizing agent, since GH increases brain ghrelin receptorexpression and parasympathetic neuronal activity. In addition,administration of ghrelin and GH in combination in aged animalssignificantly decreases proinflammatory cytokines and attenuates organinjury after LPS injection, indicating that ghrelin and its sensitizingagent GH represent a novel therapy for sepsis in the geriatricpopulation. Additional work was performed by using an established modelof polymicrobial sepsis (i.e., CLP) in the young rat. These resultsdemonstrate that administration of ghrelin in sepsis significantlyreduced proinflammatory cytokine levels, maintained cardiovascularstability, attenuated tissue injury, and improved survival in sepsis inyoung animals. Unlike aged animals, young animals favorably respond toghrelin treatment.

Human Ghrelin Ameliorates Organ Injury and Improves Survival afterRadiation Injury Combined with Severe Sepsis:

In the terrorist radiation exposure scenario, radiation victims likelysuffer from additional injuries such as sepsis. Despite advances inunderstanding of radiation injury and the management of septic patients,little information is available regarding radiation combined injury(RCI, e.g., radiation exposure followed by sepsis).

Rats were exposed to 5-Gy whole body irradiation followed by cecalligation and puncture (CLP) 48 h thereafter. Human ghrelin (30 nmol) orvehicle (saline) was infused for 68 h after radiation exposure. At 20 hpost-CLP, circulating tissue injury markers were measured. A 10-daysurvival study was also performed.

Human ghrelin significantly decreased the elevated injury markers inplasma by 41-59% (Table 1). Furthermore, ghrelin also significantlyincreased survival from 38% to 69% (FIG. 17). Since human ghrelinreduces organ injury and improves survival after RCI, it may serve as anovel and effective therapy for humans under radiation exposures.

TABLE 1 Ghrelin treatment decreases injury markers in plasma followingradiation exposure and sepsis (RCI). AST ALT Lactate Creatinine LDH IL-6TNF-α Gut MPO (IU/L) (IU/L) (mg/dL) (mg/dL) (U/L) (pg/ml) (pg/ml)(μU/mg) Sham 25.6 ± 1.4 22.1 ± 2.4  2.2 ± 0.3 0.38 ± 0.07  501 ± 49  26± 3  42.4 ± 11.2 0.8 ± 0.2 RCI + Vehicle 153.5 ± 7.2* 72.0 ± 2.6* 28.5 ±3.0* 2.69 ± 0.13*  2541 ± 136* 1400 ± 144* 84.2 ± 3.2* 33.8 ± 7.5* RCI +Ghrelin   70.7 ± 70.3*#  42.3 ± 5.4*#  19.4 ± 0.8*# 1.60 ± 0.20*# 1098 ±88*#  817 ± 227*# 41.8 ± 8.5#  14.0 ± 1.1*# Mean ± SE, n = 4-6/group;ANOVA & Student-Newman-Keuls; *P < 0.05 vs. Sham; #P < 0.05 vs. RCI +Vehicle. LDH—lactate dehydrogenase, MPO—myeloperoxidase.

REFERENCES

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1. A method for treating sepsis and/or septic shock in a human subjectat least 60 years of age, the method comprising administering to thesubject a combination of a ghrelin and a growth hormone in amountseffective to treat sepsis and/or septic shock.
 2. The method of claim 1,wherein ghrelin has an amino acid sequence at least 90% identical to SEQID NO:1.
 3. The method of claim 1, wherein growth hormone has an aminoacid sequence at least 90% identical to SEQ ID NO:2, SEQ ID NO:3 or SEQID NO:4.
 4. The method of claim 1, wherein ghrelin has an amino acidsequence at least 90% identical to SEQ ID NO:1, and wherein growthhormone has an amino acid sequence at least 90% identical to SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:4.
 5. The method of claim 1, whereinco-administering ghrelin and growth hormone to the subject prevents orreduces tissue and/or organ injury in the subject.
 6. The method ofclaim 1, wherein co-administering ghrelin and growth hormone to thesubject prevents or reduces serum elevation of one or more of TNF-α,interleukin-6, aspartate aminotransferase, alanine aminotransferase,bilirubin, lactate, creatinine, or high-mobility group box 1 protein. 7.The method of claim 1, wherein co-administering ghrelin and growthhormone to the subject prevents or reduces elevation of serum TNF-α. 8.The method of claim 1, wherein co-administering ghrelin and growthhormone to the subject prevents or reduces septic shock.
 9. The methodof claim 1, wherein co-administering ghrelin and growth hormone to thesubject improves cardiovascular stability, as measured by one or more ofimproved cardiac output, stroke volume, total peripheral resistance, orblood flow.
 10. The method of claim 1, wherein co-administering ghrelinand growth hormone to the subject improves pulmonary edema.
 11. Themethod of claim 1, wherein co-administering ghrelin and growth hormoneto the subject improves survival of the subject. 12-13. (canceled) 14.The method of claim 1, wherein the subject is a human at least 65 yearsof age.
 15. A pharmaceutical composition comprising ghrelin and growthhormone formulated in dosage form for treating sepsis and/or septicshock.
 16. A method of preparing a pharmaceutical composition fortreating sepsis and/or septic shock, the method comprising formulatingghrelin and growth hormone together in a pharmaceutical composition inamounts effective to treat sepsis and/or septic shock.
 17. A method forreducing organ and/or tissue injury in a subject and/or improvingsurvival of the subject after radiation exposure and sepsis comprisingadministering to the subject ghrelin in an amount effective to reduceorgan and/or tissue injury and/or improve survival, whereinadministration of ghrelin reduces serum elevation of one or more ofTNF-α, interleukin-6, aspartate aminotransferase, alanineaminotransferase, lactate, creatinine, lactate dehydrogenase ormyeloperoxidase. 18-20. (canceled)
 21. A method for treating aninflammatory condition associated with serum elevation of one or more ofTNF-α, interleukin-6, aspartate aminotransferase, or alanineaminotransferase in a human subject at least 60 years of age, the methodcomprising administering to the subject a combination of a ghrelin and agrowth hormone in amounts effective to treat the inflammatory condition.22. A method for reducing a physiological effect of sepsis in a subjectcomprising co-administering to the subject a ghrelin and a growthhormone as a ghrelin sensitizer to reduce a physiological effect ofsepsis, wherein the growth hormone is administered in an amounteffective to enhance ghrelin therapy.
 23. A method for treating aninflammatory condition in a subject associated with serum elevation ofone or more of TNF-α, interleukin-6, aspartate aminotransferase, oralanine aminotransferase, comprising co-administering to the subject aghrelin and a growth hormone as a ghrelin sensitizer to treat theinflammatory condition, wherein the growth hormone is administered in anamount effective to enhance ghrelin therapy.
 24. The method of claim 22,wherein co-administering ghrelin and growth hormone to the subjectreduces serum levels of one or more of TNF-α, interleukin-6, aspartateaminotransferase and alanine aminotransferase.
 25. The method of claim23, wherein co-administering ghrelin and growth hormone to the subjectreduces serum levels of one or more of TNF-α, interleukin-6, aspartateaminotransferase and alanine aminotransferase.